Hemocytes synthesize components of the mosquito complement-like system and are major players of the priming response. However, it is not clear how they enhance antiplasmodial immunity. The fate of mosquito hemocytes following infection with Plasmodium was investigated by labeling these cells in vivo. We found that midgut nitration triggers the local release of hemocyte-derived microvesicles (HdMv) into the basal labyrinth of the midgut. Several different strategies, such as gene silencing, immune priming or systemic injection of polystyrene beads were used to either enhance or reduce HdMv release. We provide direct experimental evidence that contact of hemocytes with the nitrated midgut basal surface triggers HdMv release, and that this response is necessary for effective activation of mosquito complement. Our studies suggest that hemocyte-derived microvesicles deliver some critical factor(s) that promote activation of thioester-containing protein 1 (TEP1), a key effector of the mosquito antiplasmodial immunity. This work was published in Science Immunology. Recent experiments revealed that midgut epithelial cells produce prostaglandins when they come in direct contact with bacteria from the gut microbiota, and that this response in necessary to establish immune priming. We found that prostaglandin release attracts hemocytes to the basal surface of the midgut and increases their motility as they patrol this interphase. We have established chemotaxis assays to evaluate the migration of hemocyte-like Sua 5.1 cells in response to prostaglandins. Insects also do not have cyclooxygenases, the enzymes that catalyze the synthesis of prostaglandins in vertebrates. We are currently screening several heme peroxidases based on their expression pattern and on the effect silencing expression by dsRNA injections on prostaglandin synthesis. We have also shown that hemocyte-derived-microvesicles (HdMv) promote complement activation. An in vitro system to induce microvesicle release by Sua 5.1 cells, as they come in contact with a nitrated extracellular matrix has been established. HdMv will be purified from this culture system and their protein cargo will be determined by proteomic analysis. The surface receptor that allows Sua 5.1 cells to respond to a nitrated surface will also be characterized using far-western blots, immobilizing cell membranes under denaturing non-reducing conditions and probing for binding of nitrated BSA with an anti-nitrotyrosine monoclonal antibody. The African (NF54) and the New World (7G8) strains differ drastically in their ability to evade the immune system of A. gambiae L35 refractory mosquitoes. Immune evasion is mediated by Pfs47, but there are only four amino acid differences between the Pfs47 protein in the NF54 and 7G8 strains. Our studies indicate that single amino acid replacements in any of the four amino acids in Pfs47 that differ between the P. falciparum NF54 and 7G8 strains, completely disrupts the ability of NF54 parasites to hide from the mosquito immune system. One of these amino acid replacements had the opposite effect on A. albimanus mosquitoes, and enhanced infection. We conclude that malaria transmission involves a complex interplay between the genetic background of the parasite and the mosquito and that Pfs47 is critical in these interactions, as it mediates Plasmodium immune evasion through molecular interactions that need to be precise in some parasite/vector combinations, such as the NF54 line with the A. gambiae L35 strain. This work was published in PlosOne. The potential of Pfs47 as a transmission blocking vaccine target was explored. We were able to produced full-length recombinant Pfs47 in E. coli and developed an in-column refolding and purification protocol. The protein is immunogenic and mouse polyclonal antibodies recognize Pfs47 in the membrane of female gametocytes. We generated 14 monoclonal antibodies, but none of them had strong transmission blocking activity. We found that all of them bound to either domain 1 (D1) or domain 3 (D3), but none of them recognized the D2-domain. The D2-domain could not be expressed alone as it was toxic to all systems tested, but when we removed the disulfide bond in the D2-loop, by replacing the two cysteines by alanines, we were able to obtain large amounts of immunogenic recombinant protein in E. coli. Polyclonal antibodies to the D2-domain have strong transmission blocking activity. We generated six new monoclonal antibodies to the D2 domain, and mapped the target regions that confers strong transmission blocking activity (98-99%). We are currently testing different constructs in collaboration with Drs. Patrick Duffy and Robert Seder to design the ideal antigen for an effective transmission blocking vaccine. We are currently investigating the mechanism of action of the vaccine. Wolbachia is an intracellular bacteria that induces refractoriness to infection with arboviruses in A. aegypti mosquitoes. We identified a strain of Wolbachia infecting natural populations of A. gambiae s.l. from Mali. The presence of Wolbachia was detected by PCR using Wolbachia-specific 16S ribosomal gene primers and confirmed by sequencing. Phylogenetic analysis showed that this Wolbachia strain in A. gambiae s.l. is a sister group to a strain previously identified in the flea Ctenocephalides sp., supporting acquisition by horizontal transfer. In field-collected mosquitoes, significantly lower Wolbachia levels were observed in Plasmodium-infected mosquitoes when compared to uninfected controls. To further investigate these interactions, a laboratory colony of Wolbachia-infected A. gambiae M form (A. colluzzii) was established and females were infected with P. falciparum (NF54). We found that mosquitoes carrying high levels of Wolbachia were less likely to carry Plasmodium sporozites. The effect of Wolbachia on the oocyst stage is under evaluation. Malaria is endemic in the American continent, and the Amazonian rainforest is the region with the highest risk of transmission. The lack of suitable experimental models to infect malaria vectors from the Americas has limited the progress to understand the biology of transmission in this region. A systematic analysis of compatibility of several different Plasmodium parasite species will be used to establish a robust experimental model of malaria transmission by anopheline mosquitoes from South America. We found that Anopheles aquasalis, a major vector in coastal areas of South America, is highly refractory to infection with two strains of Plasmodium falciparum (NF54 and 7G8) and with Plasmodium berghei (mouse malaria), even when the microbiota was eliminated with antibiotics and oxidative stress was reduced by oral administration of uric acid. In contrast, An. aquasalis females are susceptible to infection with another murine parasite, Plasmodium yoelii nigeriensis N67 (PyN67). Anopheles albimanus, one of the main malaria vectors in Central America, Southern Mexico and the Caribbean, was more susceptible to infection with PyN67 than An. aquasalis, even in the absence of any pre-treatment. Disruption of the complement-like system in Anopheles albimanus significantly enhanced PyN67 infection, indicating that the mosquito immune system is mounting an effective antiplasmodial response. Plasmodium yoelii nigeriensis has the ability to infect a broad range of anophelines, and is an excellent model to study malaria transmission by South American vectors. This work was published in PlosOne.